This project responds to the NIEHS RFA-ES-20-005 ?Organotypic culture models developed from experimental animals for chemical toxicity screening,? that is aligned with the needs of the NTP?s Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) for development and evaluation of new, revised, and alternative methods to identify potential hazards to human health and the environment. Through our ongoing NCATS SBIR Phase II, Lena Biosciences (LB) developed and commercialized an organ- on-a-chip-like, Perfused Organ Panel, with a proprietary liquid breathing technology that provides uniform interstitial perfusion, superior delivery of oxygen and stable pH to 48 statistically independent organ cultures. In this project, Lena Biosciences will use the Perfused Organ Panel to develop physiologically-relevant in vitro screening systems using cells derived from animal species typically utilized for toxicological testing. Next, LB will replicate biological interactions and toxicological responses observed in animal tissues. Lastly, LB will produce assay data that is suitable for comparisons between in vitro and in vivo animal toxicology studies, and Tox21 HTS data. Ultimately, this project will provide thoroughly characterized and validated, alternative in vitro test systems with high specificity and sensitivity to reduce or replace the use of animals in toxicity testing. In Phase I, LB will develop biologically and xenobiotic-metabolically competent rodent liver and brain models having in vivo like cellular respiratory metabolism to achieve optimal mitochondrial responsiveness and susceptibility to toxicants. The liver model will provide high activity of drug metabolizing enzymes for in situ generation of reactive metabolites, and mimic parallel processes of parent drug deletion and metabolite formation to better model and predict toxicological outcomes. The brain model(s) will mimic the brain?s innate immunity with robust toxicological responses to drug overdose, Acetylcholinesterase inhibition with acute neurotoxicity (Phase I), and neuropathy target esterase inhibition that cause delayed neuropathy (Phase II) following the exposure to organophosphorus (OP) chemicals and their toxic metabolites. Perfused Organ Panel and 3 sets of in vitro controls will be treated with Acetaminophen (APAP) and Malathion, an OP insecticide with a neurotoxic metabolite, Malaoxon, that are relevant to the testing of specific tissue models and for which species-matched in vivo data already exists. This will facilitate benchmarking and show the Perfused Organ Panel?s utility as an alternative to in vivo models currently used by the U.S. Environmental Protection Agency. To successfully carry out these studies and ensure the project?s success, we assembled a team of experts in advanced cell culture models of liver and brain (LB?s PI and CSO, Dr. Shoemaker), drug metabolism and metabolite formation (Dr. Morgan, Emory, Department of Pharmacology and Chemical Biology), APAP toxicity (Dr. Jaeschke, University of Kansas Medical Center), and brain and liver toxicology (Dr. Caudle, Emory, Department of Environmental Health, Dr. Jaeschke, and Dr. Morgan).